Iron is an essential nutrient for virtually every organism, yet it can also be a potent cellular toxin. Dysregulated iron metabolism and iron overload are features of a growing number of human diseases. Some genes involved in cellular iron uptake and export have been identified, yet very little is known about inter- and intracellular iron transport, intracellular iron utilization, and the regulation of these processes. A combination of genetic, biochemical, and cell biological approaches is needed to understand iron metabolism and the role of iron in human disease. These approaches can be combined in the simple eukaryote, Saccharomyces cerevisiae. Studies of metal metabolism in budding yeast have yielded important insights into iron, copper, and zinc metabolism in both humans and pathogenic microorganisms. Genetic studies of iron metabolism in a simple eukaryote will allow us to discover new genes involved in iron homeostasis as well as to determine the cellular response to iron overload and iron deprivation. We have used cDNA microarrays representing the entire yeast genome to identify genes that are regulated according to the availability of iron and the activity of Aft1p, the major iron-dependent transcription factor. Using available genome and protein databases, we have grouped these newly identified genes into families and have begun their functional evaluation. Three genes that are predicted to encode GPI-anchored proteins of the yeast cell wall are new members of the Aft1 regulon. The corresponding mRNA transcripts show over a 50-fold induction under conditions of iron deprivation and Aft1-1up expression and the upstream control regions of the genes contain multiple Aft1p consensus sites. We confirmed that these proteins were incorporated into the cell wall by immunofluorescence. Strains bearing deletions of these genes were constructed and they exhibit defects in the retention of iron in the cell wall. Additionally, these strains exhibit reduced rates of siderophore iron uptake and evidence of altered intracellular iron sensing, indicating that cell wall mannoproteins contribute to iron uptake at the plasma membrane. We have identified and genetically characterized a novel system of eukaryotic iron uptake. Four homologous genes regulated as part of the Aft1-regulon (ARN1-4) were found to facilitate the transport of siderophores. We are investigating how the Arn proteins facilitate the uptake of siderophore when they do not appear to be expressed on the plasma membrane. To track the path of Arn proteins within the cell, we have examined siderophore uptake and Arn protein localization in a series of well-characterized yeast mutants that exhibit temperature-sensitive defects in protein sorting. These experiments are revealing that, in the absence of transport substrate, Arn1p is sorted directly from the Golgi to the late endosome/pre-vacuolar compartment and does not cycle to the plasma membrane. Upon the addition of specific substrate, however, Arn1p begins cycling to the plasma membrane by a process of regulated exocytosis. This regulated exocytosis occurs only in response to the specific substrate, other siderophores that are not substrates do not elicit translocation. Our data suggest that siderophore binding at the plasma membrane and siderophore transport across the endosomal membrane may be separate and necessary steps in siderophore uptake. Because siderophore iron uptake may be important for virulence in pathogenic fungi, we have entered into a collaboration with the laboratory of Dr. Jerry Kaplan to identify siderophore transporters in Candida albicans. Using strains of S. cerevisiae developed in our laboratory, the C. albicans ferrichrome transporter was cloned and functionally characterized. We explored the role of surface reductases in the uptake of siderophores through the plasma membrane-based system. Uptake through the plasma membrane ferrous iron transporter requires that the iron first dissociate from the siderophore and undergo reduction to the ferrous form. FRE1 and FRE2 encode cell surface metalloreductases that are required for reduction and uptake of free ferric iron. The yeast genome contains five additional FRE1 and FRE2 homologues, four of which are regulated by iron and Aft1p, but their function was unknown. Fre3p was required for the reduction and uptake of ferrioxamine B-iron and for growth on ferrioxamine B, ferrichrome, triacetylfusarinine C, and rhodotorulic acid in the absence of Fre1p and Fre2p. Fre4p could facilitate utilization of rhodotorulic acid-iron when the siderophore was present in higher concentrations. We proposed that Fre3p and Fre4p are siderophore?iron reductases, and that the apparent redundancy of the FRE genes confers the capacity to utilize iron from a variety of siderophore sources. We plan to continue our functional analysis of new members of the Aft1 regulon and to investigate the cellular response to iron deprivation and iron overload. Our aim is to extend this work to examine analogous processes in human cells.